Our studies are focused on examination of single islet gene expression profiles (transcriptomes) to determine pathways critical to human beta cell death and survival. In preliminary studies we have developed new approaches and protocols that allow laser-capture of individual islets from cryosections of human pancreas, extract the RNA, and obtain comprehensive transcriptomes. Our proposal to the HIRN-CBDS consortium is based on using and expanding these proven protocols to gain deeper insights into islet heterogeneity and differences in gene expression patterns of individual islets from individuals with and without diabetes and with or without serum autoantibodies (AAB). Based on preliminary data in islets from non-diabetic individuals with or without AAB, we hypothesize that islets develop a gene expression signature of cellular stress before the early stages of insuliti. A full characterization of early abnormalities in islet gene expression signatures could point to novel mechanisms of disease and new therapeutic strategies that target islet abnormalities to delay or prevent beta cell death. The use of comprehensive gene expression profiling of thousands of expressed genes will allow a very detailed look at specific pathways and processes. Our proposal has the following specific aims: Specific Aim 1. To characterize transcriptomes of individual islets in a survey of islets from different regions of the pancreas an from organ donors with or without autoantibodies (AAB) and with or without diabetes. This will provide an unbiased survey of islet gene expression and its heterogeneity. Specific Aim 2. To characterize transcriptomes of individual islets selected by rapid immuno-laser capture microdissection for the presence or absence of insulin-positive beta cells and presence or absence of CD3 T-cells. In this aim we will obtain expression profiles from islets that have been selected based on multiple fluorescent stains for beta cells and insulitis. We will develop protocols for using rapid immunofluorescence that provide high quality RNA and then directly microdissect islets, across serial sections, based on their staining patterns. Specific Aim 3. To characterize transcriptomes of individual islets selected for staining patterns of markers of viral presence and metabolic stress. In this aim we will use our newly acquired ability to conduct multiplexed immunofluorescence to define islets with novel complex phenotypes indicative of stress due to viral infections or metabolic stress. In this aim phenotypes will be highly complex and we will stain one tissue section and then capture individual islets of interest from adjacent serial tissue sections. Mechanistic studies using isolated islets will follow from all specific aim as new pathways of cellular destruction or survival are discovered.